The TCR:CD3 complex is one of the most important receptors in the immune system. The central theme and long-term objective of this project is to 'determine how TCR:CD3 complexity mediates the multitude of downstream functional events orchestrated by different T cell subsets.'The TCR:CD3 complex is unique in having ten ITAMs, but the physiological significance of this is unclear. In addition to the 23 publications contributed to by this project, we have developed a powerful approach to dissect the contribution of CD3 ITAMs in mediating T cell development and function, which combines retroviral-mediated stem cell gene transfer and 2A peptide-linked multicistronic retroviral vectors. This allowed us to generate 25 groups of 'retrogenic'mice expressing different combinations of wild type and ITAM mutant TCR:CD3 complexes. Our results show that some CD3 mutants cause a lethal, rapidly-developing multiorgan disease. We also found dramatic differences in the TCR signal required for induction of proliferation versus cytokine production. These observations led to the hypothesis that the 'TCR:CD3 complex has the unique ability to provide scalable signaling with a broad dynamic range that can differentially modulate distinct T cell parameters'. In this project, we will use our unique CD3 retrogenic approach, advanced microscopy [time-lapse spinning disk confocal microscopy, with wide-field deconvolution, and total internal reflected fluorescent microscopy (TIRFM) with T cell:APC couples and anti-TCRb/ICAM or MHC class II/ICAM supported lipid bilayers], multiplexed, reverse-phase protein lysate (RPPL) microarrays, and conventional biochemical and functional assays. AIM 1: The mechanistic basis for the differential CD3 ITAM requirement for CD4+ T cell proliferation and cytokine production. (A) How does CD3 ITAM number affect IS assembly, kinetics and stability? (B) How does CD3 ITAM number affect the initiation and transmission of TCR signaling? (C) How does CD3 ITAM number affect the mechanism of cytokine production? AIM 2: The relative contribution of CD3 ITAM flavor and location in mediating TCR signaling and T cell function. Our preliminary studies suggest that CD3 ITAM diversity is important as mice expressing CD3 complexes that contain a single ITAM 'flavor'(eg. all ITAMs mutated to the same ITAM sequence) exhibit profound defects in T cell function. (A) What is the consequence of limiting CD3 ITAM diversity on T cell function? (B) What is the consequence of limiting CD3 ITAM diversity on TCR signaling? (C) How important is the location of CD3 ITAMs within the TCR complex in mediating signaling and T cell function? AIM 3: CD3 ITAM requirements for Treg signaling and function. (A) Is Treg function subverted in mice with altered CD3 ITAM number, flavor and/or location? (B) Do alterations in CD3 ITAM number, flavor and/or location have the same effect on IS mechanics and TCR signaling in Tregs?